Orientador: Elizabete Jordão / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-23T06:39:10Z (GMT). No. of bitstreams: 1 Paschoalino_FlaviaCristinaSertori_D.pdf: 2972694 bytes, checksum: 77c5309ec8bdff08525a02274c840aae (MD5) Previous issue date: 2013 / Resumo: Este estudo apresenta a aplicação de três diferentes sistemas fotocatalíticos: (1) reator vítreo com fonte de radiação UV-C (250 W) operando em modo batelada com recirculação, cujo objetivo foi avaliar o desempenho dos fotocatalisadores TiO2, ZnO, CuO ou Ga2O3 em suspensão na degradação de uma solução de fenol a 100 mg L-1. Os resultados mostraram que quando empregado TiO2 P25 foi possível mineralizar 80% do fenol em solução após 150 min, sendo necessários 300 min para obter o mesmo desempenho com ZnO. Os óxidos CuO e Ga2O3 não apresentaram degradação fotocatalítica significativa; (2) reator solar tipo coletor parabólico composto (CPC) operando em modo contínuo ou batelada, com a finalidade de avaliar uma superfície suportada de TiO2 P25 sobre um óxido misto sol-gel TiO2/SiO2 na degradação de antibióticos (levofloxacina, ciprofloxacina, enrofloxacina, trimetoprim) em concentrações comumente encontradas em amostras ambientais. Os compostos ciprofloxacina e enrofloxacina foram rapidamente degradados por fotólise direta enquanto que a levofloxacina e o trimetoprim apresentaram-se mais recalcitrantes, sendo possível atingir valores de degradação próximos a 80% para a levofloxacina e 40% para o trimetoprim; (3) reator construído em Ti com fonte de radiação UV-C (102 W) operando em modo batelada com recirculação, cujo objetivo foi avaliar o desempenho do fotocatalisador TiO2 P25 em suspensão em conjunto com H2O2 na oxidação de uma solução de glicerol 100 mmol L-1 visando a obtenção de substâncias de alto valor agregado. Utilizou-se de um planejamento fatorial para avaliar a influência da concentração inicial do glicerol, do TiO2 e do H2O2. Alcançaram-se valores de degradação máxima do glicerol ao redor de 50%, sendo que o intermediário de degradação identificado de maior valor agregado foi a 1,3-dihidroxiacetona (DHA) com seletividade máxima de 15% / Abstract: This study presents the application of three different photocatalytic systems: (1) glass made reactor using an UV-C source (250 W) operating in batch mode through recirculation with the purpose to evaluate the performance of the photocatalysts TiO2, ZnO, CuO or Ga2O3 in suspension on the degradation of a phenol 100 mg L-1 aqueous solution. Results shown that when using TiO2 P25 it was possible to mineralize 80% of phenol after 150 min reaction. When using ZnO it was necessary 300 min reaction to achieve the same performance. CuO and Ga2O3 did not present significant efficiency; (2) compound parabolic collector (CPC) solar reactor operating in batch or continuous mode with the purpose of evaluate a TiO2 P25 surface supported onto a TiO2/SiO2 layer on the degradation of antibiotics (levofloxacyn, ciprofloxacyn, enrofloxacyn, trimetoprym) under common concentrations of environmental water samples. Ciprofloxacyn and enrofloxacyn were fastly degraded by direct photolysis process while levofloxacyn and trimetoprym presented more recalcitrant. It was possible to reach 80% degradation for levofloxacyn and 40% for trimetoprym; (3) titanium assembled reactor using an UV-C source (102 W) operating in batch mode under recirculation with the purpose of evaluate TiO2 P25 aqueous suspension in conjunction with H2O2 on the generation of substances with high commercial value. It was used a factorial planning to evaluate the influence of glycerol, TiO2 and H2O2 initial concentration. It was possible to obtain glycerol degradation of 50% and the main intermediate identified was 1,3-dihydroxyacetone (DHA) with a maximum selectivity of 15% / Doutorado / Sistemas de Processos Quimicos e Informatica / Doutora em Engenharia Quimica

Orientador: José Roberto Guimarães / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Civil, Arquitetura e Urbanismo / Made available in DSpace on 2018-08-27T20:18:28Z (GMT). No. of bitstreams: 1 Oliveira_AmandaMarchiDuartede_M.pdf: 2576445 bytes, checksum: b72f8b00f5d7ccd5b5b4282cff3876ce (MD5) Previous issue date: 2015 / Resumo: O antimicrobiano lomefloxacina (LOM) foi detectado em matrizes aquosas em vários países. Sua introdução contínua no ambiente pode apresentar risco potencial para organismos aquáticos e terrestres, devido à presença de atividade antimicrobiana. A fim de degradar a LOM e outros contaminantes emergentes, utiliza-se os processos oxidativos avançados (UV/H2O2, O3 em pH 11) devido à geração de radicais hidroxila altamente reativos e não seletivos. O objetivo deste estudo foi avaliar a degradação da LOM por fotólise, peroxidação, peroxidação assistida por radiação ultravioleta e ozonização em diferentes valores de pH, além de avaliar a atividade antimicrobiana residual, a toxicidade aguda e também identificar e sugerir os produtos formados durante os processos de degradação. Soluções de 500 ?g L-1 de LOM foram submetidas aos processos fotolítico (? = 254 nm), peroxidação (CH2O2 = 0,335; 1,675 e 6,77 mmol L-1); UV/H2O2 (CH2O2 de 0,067 a 6,77 mmol L-1) e ozonização (Dose O3 = 5,5 a 54,34 mg L-1) em diferentes valores de pH (3, 7 e 11). Após ensaio de degradação em escala laboratorial, realizou-se a extração em fase sólida e injeção em HPLC para avaliar o percentual de degradação. Foram avaliadas também a remoção da atividade antimicrobiana, com o organismo-teste Escherichia coli K12 (ATCC 23716), e a toxicidade com o organismo-teste Vibrio fischeri (Microtox). Com os resultados do presente trabalho foi possível sugerir que a peroxidação foi ineficaz para a degradação da lomefloxacina: menos que 15% da concentração do fármaco foi reduzida, mesmo após rtempo de ensaio (60 minutos); esse processo também foi ineficaz na redução da atividade antimicrobiana. Ambos os processos de fotólise e UV/H2O2 foram capazes de degradar mais de 95% de LOM em 60 minutos (Dose de radiação = 5267 mJ cm-2). Em relação à degradação da LOM, não se observou diferença quando utilizado UV (95,7%) ou UV/H2O2 (concentração de H2O2 variando de 0,067 a 6,77 mmol L-1) (96,9%). No entanto, o processo UV/H2O2 com 6,77 mmol L-1 de H2O2 foi o mais eficaz na redução da atividade antimicrobiana (96,3%). Em todos os processos avaliados, não foi verificado a remoção completa da atividade antimicrobiana. A ozonização em pH 3 apresentou eficiência quanto à degradação, redução da atividade antimicrobiana e, principalmente da toxicidade aguda, sendo a inibição da V. fischeri de 12,8%. Em todos os processos observou-se incremento na toxicidade, sugerindo a formação de produtos intermediários de degradação mais tóxicos que o composto original. Os processos avaliados demonstraram alto potencial para serem incorporados ou utilizados em estações de tratamento de água que contenham a LOM e/ou compostos químicos emergentes / Abstract: The antimicrobial drug lomefloxacin (LOM) has been detected in the aqueous matrices in several countries. Its continuous introduction into the environment is a potential risk to aquatic and terrestrial organisms due to the presence of antimicrobial activity. Advanced oxidation processes (UV/H2O2, O3 pH 11) due to hydroxyl radicals that were highly reactive and non-selective were used to degrade emerging contaminants and were evaluated to degraded LOM aqueous solutions. The aim of this study was to evaluate the degradation of LOM by photolysis, peroxidation, peroxidation assisted by ultraviolet radiation and ozonation at different pH values; moreover, the residual antimicrobial activity, the acute toxicity, and the degradation byproducts formed during degradation processes were evaluated. Aqueous solutions of 500 ?g L-1 LOM were underwent to UV radiation (? = 254 nm), peroxidation (CH2O2 = 0.335; 1.675 e 6.77 mmol L-1), UV/H2O2 (CH2O2 0.067 to 6.77 mmol L-1) and the ozonation (Dose O3 = 5.5 to 54.34 mg L-1) at different pH values (3, 7 and 11). LOM degradation was evaluated by high performance liquid chromatography (HPLC) with UV detection. Samples were prepared prior to measurements by solid phase extraction (SPE). Antimicrobial activity was determined by evaluating growth inhibition of Escherichia coli K12 (ATCC 23716) and the acute toxicity of Vibrio fischeri test organism (Microtox). Peroxidation did not effectively degrade LOM, reducing less than 15% of the initial drug concentration (500 ?g L-1) after 60 min of testing; this process was also ineffective to reduce antimicrobial activity. Both photolysis and UV/H2O2 (6.7 10-2 mmol L-1 H2O2) were able to degrade more than 95% of LOM in 60 min of testing (radiation dose = 5267 mJ cm-2). However, the UV/ H2O2 (CH2O2 = 6.77 mmol L-1) was more effective to reduce the antimicrobial activity (96.3%). Antimicrobial activity of the solutions was not totally eliminated in any of the processes evaluated. The ozonation at pH 3 was efficient to degradation, reduction of antimicrobial activity and, especially of acute toxicity, with an inhibition of 12.8% of V. fischeri. In all cases there was an increase in toxicity, suggesting the formation of toxic intermediates more degradation than the parent compound. The evaluated processes showed high potential to be incorporated or used in water treatment plants that contain the LOM and/or emerging contaminants / Mestrado / Saneamento e Ambiente / Mestra em Engenharia Civil

Águas residuárias são continuamente contaminadas por fármacos. Dentre estes fármacos, os antimicrobianos causam grande preocupação pelos impactos sobre o desenvolvimento de resistência bacteriana. As principais fontes de contaminação destes fármacos são efluentes urbanos, hospitalares, de fazendas e de algumas indústrias. A complexidade das matrizes ambientais tais como águas residuárias é uma das principais dificuldades para extrair e detectar fármacos, fazendo-se necessário o uso de técnicas de preparo de amostra para a extração destes compostos de interesse. Técnicas clássicas como a extração líquido-líquido (LLE) e a extração em fase sólida (SPE) são largamente usadas para extração de fármacos nesse tipo de matriz, porém estas técnicas não atendem amplamente aos princípios da química verde. Dessa forma, novas técnicas, mais alinhadas à responsabilidade ambiental, têm sido desenvolvidas. Neste âmbito apresenta-se o desenvolvimento e a validação de um método de microextração líquido-líquido para extração e detecção de sulfonamidas e o desenvolvimento e otimização de um método utilizando planejamento experimental para a extração de fluoroquinolonas em águas residuárias. Foi possível obter-se o limite de detecção de 0,2 ng mL-1 para as sulfonamidas analisadas, este LD é relativamente baixo considerando que o detector que foi utilizado não possuía a possibilidade de fazer análises no modo MS/MS, o que certamente reduziria ainda mais o LD. Com os desenvolvimentos desse trabalho tornou-se possível a utilização de apenas 1 mL de solvente orgânico para a pré-concentração off-line, Esta etapa, adicionada a uma outra pré-concentração online (column switching) permitiu a extração dos analitos com a obtenção de um LD relativamente baixo, a partir de apenas 7 mL de amostra. / Drugs are continuously contaminating wastewater. Among these drugs antimicrobials cause great concern for the impacts on the development of bacterial resistance. The main sources of contamination by these drugs are urban effluents, hospitals, farms and some industries. The complexity of the environmental matrices such as wastewater is one of the main difficulties in extracting and detecting drugs, bringing up the need to use sample preparation techniques for the extraction of the interest compounds. Classical techniques such as liquid-liquid extraction (LLE) and solid phase extraction (SPE) are widely used for drug extraction in this type of matrix, but these techniques do not largely meet the principles of green chemistry. In this way, new techniques, more aligned with environmental responsibility, have been developed. In this context, this thesis presents the development and validation of a liquid-liquid microextraction method for sulfonamide extraction and detection and the development and optimization of a method using experimental design for the extraction of fluoroquinolones presented in wastewater. It was possible to obtain a limit of detection (LD) of 0.2 ng mL-1 for the sulfonamides analyzed, this LD is relatively low considering that the detector that was used did not have the possibility to perform analyzes in the MS/MS mode, which certainly would further reduce the LD. With the development of this thesis, it became possible to use only 1 mL of organic solvent for the off-line preconcentration of the analytes. This step, added to another online preconcentration (column switching) allowed the extraction of the analytes obtaining relatively low LDs, from just 7 mL of the sample.

This master‘s thesis is focused on the optimization of sample preparation and analytical method for the determination of fluoroquinolone. The theoretical part describes the principles of analysis in soil samples, environmental aspects of antibiotic resistance, characteristics of UPLC-MS / MS methods, description of quinolone. The experimental part deals with the identification and determination of fluoroquinolone substance (ciprofloxacin, enrofloxacin) in soil in which manure from poultry litter was incorporated using liquid chromatography with mass spectrometry (MS / MS, triple quadrupole). Internal standard and standard addition methods were used to quantify ciprofloxacin and enrofloxacin. The amount of CIP in the samples ranged from 0.2030 to 12.7088 µg·g-1 and for ENR from 0.2537 to 10.0224 µg·g-1.

The diploma thesis is focused on the determination of tetracyclines, sulfonamides and fluoroquinolones from soil. Antibiotics such as tetracycline, chlortetracycline, oxytetracycline. ciprofloxacin, trimethoprim, sulfamethaxazole and sulfadiazin were selected due to frequent use in veterinary medicine. Due to the fact that the soil is a complex matrix and contains many components that can interfere with the detection signal of analytes, it was necessary to use the MAX column in the SPE method, which removes fulvic aned humic acid from analytes and the HLB column Final analysis of the analytes was performed by liquid chromatography with mass detesction (LC-MS). The method was also used for real soil samples, which were delivered from ÚKZUS.

Polycyclic aromatic compounds (PACs), which comprise a complex class of condensed multi-ring benzenoid compounds, are important environmental pollutants originating from a wide variety of natural and anthropogenic sources. PACs are generally formed during incomplete combustion of pyrolisis of organic matter containing carbon and hydrogen. Because combustion of organic materials is involved in countless natural processes or human activities, PACs are omnipresent and abundant pollutants in air, soil, and water. Chemical analysis of PACs is of great environmental and toxicological importance. Many of them are highly suspect as etiological agents in human cancer. Because PACs carcinogenic properties strongly depend on molecular structure and differ significantly from isomer to isomer, it is of paramount importance to determine the most toxic isomers even if they are present at much lower concentrations than their less toxic isomers. Gas chromatography (GC), high-resolution GC, and high-performance liquid chromatography (HPLC) are the basis for standard PACs identification and determination. Many cases exist where GC, HPLC, and even HR-GC have not been capable to provide unambiguous isomer identification. The lack of reliable analytical data has lead to serious errors in environmental and toxicological studies. This dissertation deals with the development of novel instrumentation and analytical methods for the analysis of PACs in environmental samples. The developed methodology is based on two well-known high-resolution luminescence techniques, namely Shpol'skii Spectroscopy (SS) and Fluorescence Line Narrowing Spectroscopy (FLNS). Although these two techniques have long been recognized for their capability in providing direct determination of target PACs in complex environmental samples, several reasons have hampered their widespread use for the problem at hand. These include inconvenient sample freezing procedures; questions about signal reproducibility; lengthy spectral acquisition, which might cause severe sample degradation due to prolonged excitation; broadband fluorescence background that degrades quality of spectra, precision of measurements and detection limits; solvent constrains imposed by the need of optically transparent media; and, most importantly, the lack of selectivity and sensitivity for unambiguous determination of closely related PACs metabolites. This dissertation presents significant advances on all fronts. The analytical methodology is then extended to the analysis of fluoroquinolones (FQs) in aqueous samples. FQs are one of the most powerful classes of antibiotics currently used for the treatment of urinary tract infections. Their widespread use in both human and animal medicine has prompted their appearance in aquatic systems. The search for a universal method capable to face this new environmental challenge has been centered on HPLC. Depending on the FQ and its concentration level, successful determination has been accomplished with mass spectrometry, room-temperature fluorescence (RTF) or UV absorption spectrometry. Unfortunately, no single detection mode has shown the ability to detect all FQ at the concentration ratios found in environmental waters. We provide a feasible alternative based on FLNS. On the instrumentation side, we present a single instrument with the capability to collect multidimensional data formats in both the fluorescence and the phosphorescence time domains. We demonstrate the ability to perform luminescence measurements in highly scattering media by comparing the precision of measurements in optically transparent solvents (Shpol'skii solvents) to those obtained in "snow-like" matrixes and solid samples. For decades, conventional low-temperature methodology has been restricted to optically transparent media. This restriction has limited its application to organic solvents that freeze into a glass. In this dissertation, we remove this limitation with the use of cryogenic fiber-optic probes. Our final efforts deal with low-temperature absorption measurements. Recording absorption spectra via transmittance through frozen matrixes is a challenging task. The main reason is the difficulty to overcome the strong scattering light reaching the detector. This is particularly true when thick samples are necessary for recording absorption spectra of weak oscillators. In the case of strongly fluorescent compounds, additional errors in absorbance measurements arise from the emission reaching the detector, which might have comparable intensity to that of the transmitted light. We present a fundamentally different approach to low-temperature absorption measurements as the sought-for-information is the intensity of laser excitation returning from the frozen sample to the intensified-charge coupled device (ICCD). Laser excitation is collected with the aid of a cryogenic fiber optic probe. The feasibility of our approach is demonstrated with single-site and multiple-site Shpol'skii systems. 4.2K absorption spectra show excellent agreement to their literature counterparts recorded via transmittance with closed cycle cryogenators. Fluorescence quantum yields measured at room-temperature compare well to experimental data acquired in our lab via classical methodology. Similar agreement is observed between 77K fluorescence quantum yields and previously reported data acquired with classical methodology. We then extend our approach to generate original data on fluorescence quantum yields at 4.2K.

Yes / We investigated whether companion drug resistance was associated with adverse outcome of the shorter MDR-TB regimen in Bangladesh, after adjusting for fluoroquinolone resistance. MDR/RR-TB patients registered for treatment with a standardized gatifloxacin-based shorter MDR-TB regimen were selected for the study. Drug resistance was determined using the proportion method, gatifloxacin and isoniazid minimum inhibitory concentration testing for selected isolates, and whole genome sequencing. Low-level and high-level fluoroquinolone resistance were the most important predictors of adverse outcomes, with pyrazinamide resistance having a significant yet lower impact. In patients with fluoroquinolone-/second-line injectable-susceptible TB, non-eligibility to the shorter MDR-TB regimen (initial resistance to either pyrazinamide, ethionamide, or ethambutol) was not associated with adverse outcome (aOR 1.01; 95%CI 0.4-2.8). Kanamycin resistance was uncommon (1.3%). Increasing levels of resistance to isoniazid predicted treatment failure, also in a subgroup of patients with high-level fluoroquinolone-resistant TB. Our results suggest that resistance to companion drugs of the shorter MDR-TB regimen, except kanamycin resistance, is of no clinical importance as long as fluoroquinolone susceptibility is preserved. Hence, contrary to current WHO guidelines, exclusions to the standard regimen are justified only in the case of fluoroquinolone, and possibly kanamycin resistance. / Damien Foundation Belgium for its financial and logistic support to run the project including its research activities. European Research Council (Starting Grant INTERRUPTB 311725).

Les pompes d'efflux sont des complexes macromoléculaires qui permettent l'efflux des antibiotiques à travers les deux membranes (interne et externe). Ces pompes, spécifiques des bactéries Gram négatif, constituent l'un des acteurs majeurs du phénomène de résistance aux antibiotiques, en contribuant ainsi à la résistance intrinsèque et acquise de ces bactéries à de nombreuses molécules utilisées en antibiothérapie. Chez P. aeruginosa, ces pompes appartiennent pour la plupart à la famille des transporteurs RND. Ce sont des complexes tripartites constitués d'un transporteur de la membrane interne (RND), d'un adaptateur périplasmique (MFP) et d'un canal de la membrane externe (OMF). Ces composants ont été caractérisés individuellement chez de nombreuses bactéries. Cependant, les mécanismes qui régissent l'assemblage et la dissociation de la pompe, essentiels pour son fonctionnement, demeurent mal compris. Nous nous sommes donc intéressés au cours de ce travail aux pompes à efflux de Pseudomonas aeruginosa. Nous avons notamment procédé à la caractérisation structurale du canal OprN de la pompe MexEF-OprN impliquée dans la résistance aux fluoroquinolones et à la caractérisation biophysique du transporteur RND MexY de la pompe MexXY-OprM, spécifique des aminoglycosides. Nous avons étudié en parallèle le mécanisme d'ouverture du canal OprM seul in vitro (aspects structuraux) et in vivo (aspects fonctionnels) au sein de la pompe assemblée. Nos résultats montrent que les OMFs de P. aeruginosa présentent des similitudes avec les OMFs d'autres bactéries Gram négatif, mais également des différences, notamment pour OprN et OprM au niveau de l'interaction avec leurs partenaires ou encore pour OprM concernant l'ouverture du canal. Nous avons par ailleurs participé à l'étude in vitro de l'assemblage et du transport à travers la pompe MexAB-OprM, reconstituée au sein de protéoliposomes, confirmant l'importance de l'acylation de la MFP dans la formation du complexe et montrant l'importance de la force proto-motrice dans l'assemblage de la pompe mais pas dans sa dissociation. En parallèle de l'étude des pompes à efflux, nous nous sommes intéressés à un autre système de résistance, impliqué dans la dégradation des antibiotiques. Nous avons donc réalisé, en collaboration avec le Pr Patrick Plésiat (laboratoire de Bactériologie de Besançon), la modélisation de mutants de la beta-lactamase AmpC de P. aeruginosa, permettant de lier les effets fonctionnels observés à des hypothèses de modifications conformationnelles. / Multidrug efflux systems are membrane transport proteins that are used to translocate a wide variety of drugs across the inner and the outer membranes of Gram-negative bacteria, leading to natural and acquired antimicrobial resistances.Most of the multidrug transporters of P. aeruginosa belong to the resistance-nodulation-cell division (RND) superfamily.They function as three-component assemblies made of an inner membrane transporter (RND), a periplasmic membrane fusion protein (MFP) and an outer membrane factor channel (OMF). Along with functional studies, many crystal structures of the individual components of the pump have been solved but the interactions underlying the complex assembly and the opening mechanism of the outer channel remain unclear. In this study, we investigated structural and functional insights of P. aeruginosa efflux pumps. We solved the crystal structure of the MexEF-OprN efflux pump outer membrane channel OprN mainly involved in fluoroquinolones resistance. Our new structure highlights the differences between P. aeruginosa and other Gram-negative bacteria OMFs that could explain their specific interaction with the cognate MFP partners. We also purified and characterized the inner membrane transporter MexY from the MexXY-OprM efflux pump, which is the major determinant of aminoglycosides resistance in P. aeruginosa. Besides, we solved the crystal structure of a mutatedform of the outer membrane channel OprM in order to understand its opening mechanism. We also investigated in vivo effect of the OprM mutants in antibiotics resistance by MIC measurements and tried to correlate with the observed structural modifications leading to the open state. Moreover, we set up a new in vitro test allowing the investigation of the assembly of the MexAB-OprM efflux pump. Our results showed the importance of MexA and its lipid anchor in promoting and stabilizing the complex assembly. In addition, as a side project with the group of Pr Plésiat (laboratoire de Bactériologie de Besançon), we built different structural models of AmpC mutants from overproducing clinical isolates,showing the possible conformational changes that lead to the resistance increase.

La résistance aux antibiotiques a pris une ampleur considérable du fait de l’utilisation des antibiotiques dans de nombreux domaines. Pour respecter l’approche « OneHealth », il est essentiel d’avoir une image spécifique de chaque situation, afin d’orienter les recommandations et de limiter la dissémination des gènes, des plasmides et des clones. Nos objectifs étaient adaptés à nos populations d’étude (chevaux et porcs) afin d’ajuster les résultats aux besoins des filières. Dans la filière équine, nous avons quantifié les résistances phénotypiques et identifié les gènes de β-lactamases à spectre étendu (BLSE/AmpC) présents dans le microbiome des chevaux sains, et nous avons identifié les facteurs de risque associés à leur portage en France et au Québec. En France, nous avons également caractérisé les mécanismes de dissémination des gènes de BLSE/AmpC. Nous avons mis en évidence qu’en France et au Québec, les E. coli commensaux provenant de fèces de chevaux sains étaient majoritairement non susceptibles à l’ampicilline, l’amoxicilline/acide clavulanique et la streptomycine et que des E. coli multirésistants et porteurs de gènes codant pour des BLSE/AmpC étaient détectés dans respectivement environ 45% et 8% des chevaux. Le blaCTX-M-1 était majoritairement détecté bien qu’en France d’autres BLSE aient été identifiés (blaCTX-M-2 et blaCTX-M-14) ainsi que le gène AmpC blaCMY-2. L’administration d’un traitement médical, le nombre de personnes s’occupant des chevaux, le type d’activité et le fait de participer à un évènement équestre dans les trois derniers mois ont été identifiés comme des facteurs de risque du portage des E. coli multirésistants ou producteurs de gènes BLSE/AmpC, soit en France soit au Québec. En France, le plasmide IncHI1-ST9 était majoritairement associé aux gènes blaCTX-M-1/2 et à l’opéron fos. Pour la filière porcine, nos objectifs étaient de colliger les données de la base de données du laboratoire EcL entre 2008 et 2016, d’évaluer la présence d’un agrégat spatio-temporel pour les isolats ETEC:F4 non susceptibles à l’enrofloxacine et de caractériser ces isolats et les éléments génétiques mobiles qu’ils transportent. En effet, l’enrofloxacine est un antibiotique de haute importance en santé humaine, et doit donc faire l’objet d’une surveillance accrue. Nous avons trouvé que plus de 90% des isolats d’E. coli entérotoxinogènes détectés chez des cas de porcs malades soumis au laboratoire EcL de 2008 à 2016 au Québec, étaient multirésistants. Le virotype LT:STb:F4 prédominait jusqu’en 2014, puis a été dépassé par le virotype LT:STb:STa:F4. Un agrégat spatio-temporel d’isolats LT:STb:STa:F4 non susceptibles à l’enrofloxacine a été détecté entre 04/2015 et 09/2016 au centre de la Montérégie. Nous avons démontré la présence d’un clone ETEC:F4 non susceptible à l’enrofloxacine, à haut risque, qui se dissémine en Amérique du Nord depuis 2013. Les isolats appartenant à ce clone sont ST100, O149:H10. Ils sont multirésistants, et associés à une pathogénicité et une virulence augmentée par rapport aux isolats détectés avant 2000. Ils portent le réplicon IncFII. Les résistances et leur mécanisme de dissémination sont différents selon l’espèce animale. Ces divergences sont fonction de l’usage des antibiotiques, et des échanges possibles avec les différents protagonistes en contact avec les animaux. / Antimicrobial resistance has become an essential issue in the last decades because of the extensive use of antimicrobials in numerous sectors. In order to follow the OneHealth approach, it is critical to have a precise picture of each situation, to adjust recommendations and prevent resistance gene dissemination as well as plasmid and clone spread. Our objectives were adapted to the animal populations under study. Therefore, our results were compatible with each sector. In the equine sector, we quantified phenotypic resistance and identified β-lactamase (ESBL/AmpC) genes present in the intestinal microbiome of healthy horses and we identified risk factors associated with their carriage both in France and in Quebec. Then, in France we characterized ESBL/AmpC gene spread mechanisms. We demonstrated that commensal E. coli originating from the feces of healthy horses were mostly non-susceptible to ampicillin, amoxicillin/clavulanic acid and streptomycin. The presence of multidrug resistant E. coli and of E. coli carrying ESBL/AmpC genes was found in around 45% and 8% of horses respectively. The most frequently detected gene was blaCTX-M-1, although blaCTX-M-2 and blaCTX-M-14 were also identified in France. The AmpC gene blaCMY-2 was identified in both localities. Medical treatment, staff number, activity, and participation in an equestrian event within the last three months were identified as risk factors for MDR or ESBL/AmpC E. coli. In France, commensal E. coli from healthy horses most commonly possessed the IncHI1-ST9 plasmid. This plasmid carries blaCTX-M-1/2 genes and the fos operon. For the swine sector in Quebec, our objectives were to gather data provided by the Animal pathogenic and zoonotic E. coli (APZEC) database between 2008 and 2017, to assess the presence of a spatio-temporal cluster for enrofloxacin non-susceptible ETEC:F4 and to characterize these isolates and the mobile genetic elements they carry. Enrofloxacin is an antibiotic classified as highly important in human medicine and as such needs to come under higher scrutiny. For this sector, we demonstrated that more than 90% of enterotoxigenic E. coli (ETEC) isolates from diseased swine submitted to the EcL between 2008 and 2016, were multidrug resistant. The main virotype in 2014 was LT:STb:F4. It was subsequently replaced by the LT:STb:STa:F4 virotype. A spatio-temporal cluster of LT:STb:STa:F4 isolates non-susceptible to enrofloxacin was detected between 04/2015 and 09/2016 in the centre of the Monteregie region. These isolates constituted an ETEC:F4 high risk enrofloxacin non-susceptible clone, which has been spreading in North America since 2013. Isolates belonging to this clone are ST100, O149H10, phylogroup A, and fimH gene negative. These isolates are multidrug resistant and associated with a higher pathogenicity and virulence than isolates detected before 2000. They all carry the incFII replicon. Resistance and mechanisms of dissemination are different according to the animal species being studied. This is likely due to different patterns of antimicrobial use in each industry and possible interactions with different protagonists in contact with the animals. It is essential to understand the situation for each animal species in order to adapt recommendations for efficiently limiting the spread of resistance genes, plasmids and clones.

Nesta dissertação são apresentadas a síntese e caracterização de duas novas substâncias, consideradas aqui como metalo-fármacos em potencial: o composto iônico (C17H19FN3O3)3[RuCl6].3H2O e o complexo [Ru(C17H17FN3O3)3].4H2O, obtidos através da reação entre RuIII e a fluoroquinona ciprofloxacina em diferentes condições de síntese (meio ácido ou neutro e refluxo, respectivamente). O espectro ESI MS em modo positivo de (C17H19FN3O3)3[RuCl6].3H2O exibiu um pico principal em m/z = 994,6 que foi atribuído ao aduto (ciprofloxacina)3.H+ em fase gasosa, enquanto [Ru(C17H17FN3O3)3].4H2O apresentou picos m/z em 1093,3 e 547,1, atribuídos aos íons [Ru(C17H17FN3O3)3].H+ e [Ru(C17H17FN3O3)3].2H+, respectivamente. Os resultados de análise térmica estão em concordância com os conteúdos de água propostos para as duas substâncias. Os espectros de absorção em meio aquoso são dominados pelas transições da ciprofloxacina na região UV. Não obstante, duas bandas foram observadas em 421 e 496 nm no caso de (C17H19FN3O3)3[RuCl6].3H2O, e atribuídas à transição de transferência de carga e à transição d-d, respectivamente; no caso de [Ru(C17H17FN3O3)3].4H2O, apenas um ombro largo em torno de 450 nm foi observado na região do Visível, tendo sido atribuído à uma transição d-d. O voltamograma cíclico deste complexo exibiu um processo quase-reversível em 0,25 V (versus EPH) enquanto o composto iônico apresentou o mesmo processo em 0,11 V, sugerindo que, neste caso, o íon central de rutênio é estabilizado no estado de oxidação III através da coordenação dos átomos de oxigênio das três unidades de ciprofloxacina. O complexo de coordenação é estável por mais de 48 horas em pH fisiológico, estomacal e intestinal. Ambos foram incubados nas condições estomacais (pH = 1,5 e temperatura = 370C) e não causaram diminuições significativas nos valores de pH. / This work presents the synthesis and characterization of two potential new metallodrugs, the ionic (C17H19FN3O3)3[RuCl6].3H2O and the coordination [Ru(C17H17FN3O3)3].4H2O compounds, by combining ruthenium (III) and the fluoroquinolone ciprofloxacin in different synthetic conditions (acid media or neutral and reflux conditions, respectively). The ESI MS (positive mode) spectrum of the ionic compound displayed a main peak at m/z = 994.6, which was assigned to the gaseous phase adduct (ciprofloxacin)3.H+, while the coordination compound featured peaks at m/z 1093.3 and 547.1 ascribed to the singly and doubly charged ions [Ru(C17H17FN3O3)3].H+ and [Ru(C17H17FN3O3)3].2H+, respectively. Thermal analysis corroborated the proposed water content for both complexes. The absorption spectra of the compounds in aqueous medium are dominated by the ciprofloxacin transitions in the UV region but two bands were observed at 421nm and 496nm for the ionic compound, assigned to a charge transfer and d-d transitions. In the case of the coordination compound only a broad shoulder around 450 nm was observed in the visible region, also assigned to a ligand field transition The cyclic voltammograms of the [Ru(C17H17FN3O3)3] complex exhibited a quasi-reversible process ascribed to the Ru(II) / (III) redox pair at -0.25 V (vs SHE) while compound (C17H19FN3O3)3[RuCl6] displayed this process at -0.11V, showing that the central ruthenium ion is stabilized in the (III) oxidation state by the coordination to the hard oxygen atoms of the ciprofloxacin ligand. The coordination compound is stable under physiological, stomachal and intestinal pH over 48 hours. Both compounds are stable incubated under stomachal conditions (pH=1.5 and 37oC) without significant pH lowering.